The intracellular quality control mechanism of major histocompatibility complex class I molecules studied by controlled peptide delivery to cells - a combined cell biological, biochemical, and biophysical investigation

• Applicant: Professor Dr. Sebastian Springer
• Subject Area: Biochemistry
• Term: from 2011 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 200883174

• Final Report Year: 2016

Final Report Abstract

The project proposal was about the control of surface transport of major histocompatibility complex (MHC)2 class I molecules in the secretory pathway of mammalian cells. Class I molecules that do not manage to bind high-affinity peptides during or just after their folding in the endoplasmic reticulum (ER), also called suboptimally loaded class I molecules, are retained inside the cell, to varying degrees in different cells. Previous work by us and others suggested that suboptimally loaded class I molecules are recognized by the Golgi quality control machinery because of a biochemical feature (conformation, flexibility, dynamics) of their peptide binding site and then returned to the ER. The aim of the project was to find out what exactly the biochemical feature of suboptimally loaded class I is, and how it is read out by the cell, leading to the molecular decision to retain the protein in the early secretory pathway. We hypothesized that suboptimally loaded class I molecules are recognized by the ER-Golgi enzyme UDP-glucose:glycoprotein glucosyltransferase (UGT1 or UGGT) and modified by monoglucosylation, and that the chaperone protein calreticulin was binding to them to retain them. In the course of the project, we thoroughly investigated the role of the ends of the peptide in stabilizing the structure of class I, and we investigated the mechanisms by which class I ER export and surface transport are controlled. We made a number of unexpected observations that put the class I light chain, beta-2 microglobulin (b2m), into the center of attention. At the same time, we became skeptical of the suggested role of UGGT, and we were unable to deliver peptide to the cells as we had proposed to do. We performed a series of additional approaches such as pulse-chases, molecular dynamics simulations, and mutagenesis of class I itself, synergizing with other projects in the group.

Publications

• Tapasin dependence of MHC class I molecules correlates with their conformational flexibility. FASEB J. 25 (2011), p. 3989-98
  Malgorzata A. Garstka, Susanne Fritzsche, Isabel Lenart, Zeynep Hein, Gytis Jankevicius, Louise H. Boyle, Tim Elliott, John Trowsdale, Antony N. Antoniou, Martin Zacharias, and Sebastian Springer
  
• Investigating MHC class I folding and trafficking with pulse-chase experiments. Mol. Immunol. 55 (2013), p. 126-130
  Susanne Fritzsche and Sebastian Springer
  
• Not all empty MHC class I molecules are molten globules: Tryptophan fluorescence reveals a two-step mechanism of thermal denaturation. Mol. Immunol. 54 (2013), p. 386-396
  Saini, Sunil Kumar; Abualrous, Esam Tolba; Tigan, Anca-Sarmiza; Covella, Kathryn; Wellbrock, Ursula & Springer, Sebastian
  
• Peptide-independent stabilization of MHC class I molecules breaches cellular quality control. J. CellSci. 127 (2014), 2885-2897
  Hein, Zeynep; Uchtenhagen, Hannes; Abualrous, Esam Tolba; Saini, Sunil Kumar; Janßen, Linda; Van Hateren, Andy; Wiek, Constanze; Hanenberg, Helmut; Momburg, Frank; Achour, Adnane; Elliott, Tim; Springer, Sebastian & Boulanger, Denise
  
• Pulse-chase analysis for studying protein synthesis and maturation. (Methods article.) Curr. Protocols Prot. Sci. 78 (2014), 78:30.3.1.-30.3.23
  Fritzsche, Susanne & Springer, Sebastian
  
• Allotype-specific release from endoplasmic reticulum matrix proteins controls cell surface transport of MHC class I molecules. Traffic 16 (2015), p. 591-603
  Fritzsche, Susanne; Abualrous, Esam T.; Borchert, Britta; Momburg, Frank & Springer, Sebastian
  
• F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins. Eur. J. Immunol. 45 (2015), p. 1248-57
  Abualrous, Esam T.; Fritzsche, Susanne; Hein, Zeynep; Al‐Balushi, Mohammed S.; Reinink, Peter; Boyle, Louise H.; Wellbrock, Ursula; Antoniou, Antony N. & Springer, Sebastian
  
• The carboxy terminus of the ligand peptide determines the stability of the MHC class I molecule H-2Kb: a combined molecular dynamics and experimental study. PLoS ONE 10 (2015): e0135421
  Abualrous, Esam Tolba; Saini, Sunil Kumar; Ramnarayan, Venkat Raman; Ilca, Florin Tudor; Zacharias, Martin & Springer, Sebastian
  
• Transport and quality control of MHC class I molecules in the early secretory pathway. (Review.) Curr. Opin. Immunol. 34 (2015), 83-90
  Springer, Sebastian
  
• Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface. PLoS ONE 13(8): e0200811, 2018
  Hein, Zeynep; Borchert, Britta; Tolba Abualrous, Esam & Springer, Sebastian